Process for anaerobic oxidation of methane

ABSTRACT

Methane can be used as a source for producing hydrogen or hydrogen equivalents in a biological process using mesophilic or thermophilic bacteria of the order of the Thermotogales, e.g.  Thermotoga maritima . Hydrogen can be produced as such, or as a reducing equivalent for the reduction of various compounds such as sulphur compounds. Especially, the methane oxidation can be used for anaerobically reducing sulphate to sulphide using methane as the predominant or sole electron donor, in coculture with sulphate-reducing micro-organisms.

FIELD OF THE INVENTION

The present invention relates to an oxygen-free biological process forconverting methane to hydrogen or hydrogen equivalents. Furthermore, theinvention relates to a biological process of reducing sulphur compoundsto sulphide.

BACKGROUND

Biological methane oxidation in the presence of oxygen is awell-established process in natural habitats and in industrialapplications. It is reported that aggregates of archaea andsulphate-reducing bacteria are capable of methane oxidation in deep-seaconditions (Boetins et al, Nature, 407: 623-626 (2000), Hoehler et al,Global Biogeochemical Cycles, 8: 451-563 (1994). However, no pure ordefined microbial cultures are known that are capable of anaerobicmethane oxidation (Orphan et al., Proc. Nat. Acad. Sci. USA (2002), 99,7663-7668). Hence, anaerobic oxidation of methane is not well-understoodand is not applied on an industrial scale. The use of archaea in anindustrial process is hardly feasible, if at all, because of theirextremely low growth rates. (?)

Removal of sulphur oxide compounds such as sulphate, sulphite, sulphurdioxide, thiosulphate and the like and elemental sulphur by anaerobicconversion to sulphide at moderate or high temperature is well known,e.g. from EP-A-0451922, WO 92/17410, WO 93/24416 and WO 98/02524. Theseprocesses usually require an electron donor (or hydrogen donor) whichcan be hydrogen, carbon monoxide or organic molecules such as alcoholsand fatty acids.

It was found recently (Balk et al., Int. J. Syst. Evol. Microbiol.(2002), 52, 1361-1368) that certain Thermotoga species are capable ofanaerobically degrading methanol, alone, in coculture withMethanothermobacter or Thermodesulfovibrio species, or in the presenceof sulphur, sulphur oxide compounds or organic sulphur compounds, suchas anthraquinone-2,6-disulphonate.

SUMMARY OF THE INVENTION

It was found according to the invention that anaerobic thermophilicbacteria of the order of the Thermotogales, especially from the speciesThermotoga, are capable of converting methane, in the absence of oxygen,to hydrogen or hydrogen equivalents. The methane carbon atom was foundto be converted to carbon dioxide and was not incorporated in thebiomass. The hydrogen produced can be used as such, or can be used toprovide hydrogen equivalents suitable for reducing various compoundse.g. sulphur compounds such as sulphate, sulphate and thiosulphate. Itwas furthermore found that hydrogen equivalents required for biologicalreduction reactions can be effectively provided by methane oxidation byanaerobic methane-oxidising bacteria. Thus the invention concerns aprocess of producing hydrogen or hydrogen equivalents by anaerobicallysubjecting methane to the activity of one or more Thermotogales species.Similarly, the invention concerns a process of anaerobic oxidisingmethane using a Thermotogales species or strain. Furthermore, theinvention concerns a process for biological reduction of chemicals suchas sulphur compounds and metals, wherein the required hydrogenequivalents are produced by subjecting methane to anaerobicmethane-oxidising bacteria.

DESCRIPTION OF THE INVENTION

The invention pertains to the anaerobic, bacterial production ofhydrogen or hydrogen equivalents. In the present context, hydrogenequivalents are understood to comprise atoms, molecules or electrons,i.e. reduction equivalents, which lower the oxidation state of asubstrate. These include e.g. acetate and formate. They are alsoreferred to as electron donors. Where the present process produceshydrogen equivalents, as distinct from (molecular) hydrogen, the processis carried out in the presence of a suitable substrate capable ofaccepting the hydrogen equivalents. Where reference is made to methane,also higher alkanes and alkenes, such as ethane, ethene, propane, etc.are contemplated.

The bacteria to be used according to the invention are anaerobicmethane-oxidising (alkane-oxidising) bacteria. These bacteria includeterrestrial and aquatic (marine) species, which can be obtained fromhydrothermal sources, oil-wells, and sometimes anaerobic thermophilicbioreactors. They can grow under a variety of environmental conditionsand, depending on the natural source and possibly adaptation processes,they can be mesophilic and/or thermophilic. Examples of suitablebacteria belong to the order of the Thermotogales, which are mostlythermophilic. A description thereof is given by Wery et al. (FEMSMicrobiol. Biol. 41, (2002) 105-114) and Reysenbach et al. (Int. J.Syst. Evol. Microbiol., 52, (2002) 685-690). The use in producinghydrogen from organic sources such as sugars is described in WO02/06503. The Thermotogales include the genera Marinitoga, Geotoga,Petrotoga, Thermotoga, Thermosipho and Fervidobacterium. They areespecially from the group containing the latter three genera. Theseinclude the species (with DSM accession numbers) Thermotoga Retina (DSM3109), Thermotoga thermarum (DSM 5069), Thermotoga hypogea (DSM 11164),Thermotoga subterranea (DSM 9912), Thermotoga elfei (DSM 9442),Thermotoga lettingae (DSM 14385), Thermosipho melanesienis (DSM 12029),Thermosipho geolei DSM 13256), Fervidobacterium islandicum (DSM 5733)and F. nodosum (DSM 5306). Most of them are available in recognisedculture collection such as DSM or ATCC, and the genome of some of them,such as Thermotoga maritima, has been sequenced (Nelson et al., Nature(1999), 399, 323-329).

The methane-oxidising bacteria may be used as a pure culture of one ofthe species or strains mentioned above or as a defined mixture withother bacteria, or as a part of a mixed culture obtained fromenvironmental samples or from bioreactors, if necessary and preferablyafter adaptation to the desired process conditions. The use of a pureculture has the advantage of allowing the process to be controlled asdesired. The invention also concerns such pure cultures as well asdefined combinations of cultures as further illustrated below.

The species to be used in the process of the invention are mesophilic orthermophilic species. The thermophilic species have their maximumactivity between 50 and 100° C., but they are generally sufficientlyactive in the mesophilic temperature range for the process to be caredout at temperatures between 30 and 50° C. as well, or even from 25° C.upwards, if necessary after adaptation. Mesophilic species have theirmaximum activity between 30 and 50° C., but axe sufficiently active from20° C. and up to e.g. 60° C. The most preferred temperatures for theprocess of the invention are between 25 and 90° C., most preferablybetween 30 and 60° C.

In an embodiment of the process of the invention, the anaerobic methaneoxidation is performed for producing molecular hydrogen. The relevanttotal reaction can be simplified as follows:CH₄+2H₂O→4H₂+CO₂The culture medium contains basic mineral medium supplemented withgrowth factors into which methane is introduced e.g. by sparging oranother method tat ensures intimate contact with the micro-organisms.The hydrogen produced can be collected e.g. using gas recirculation,wherein the gas is contacted with a selective membrane which ispermeable for hydrogen and impermeable for larger molecules includingmethane, and the remaining gas can be recirculated to themethane-oxidising reactor. Alternatively, suitable selective absorbentscan be arranged in such a manner that the gas evolving from the reactoris contacted with the absorbents. Efficient withdrawal of hydrogen fromthe reaction mixture ensures sufficient bioconversion of methane tohydrogen. The hydrogen produced can be used as a fuel or as a chemicalsynthesis agent or in biological or chemical reduction processes.

In a preferred embodiment, the anaerobic methane oxidation is performedfor reducing substrates, such as nitrate, azo compounds, inorganic andorganic sulphur compounds such as elemental sulphur, sulphate, sulphite,thiosulphate, polysulphides, anthraquinone-2,6-disulphonate, dissolvedmetals, oxidised halogen compounds, nitrate and other compounds a mustbe removed. The compounds can be present in liquid waste streams, ifappropriate after extraction from the gas stream by scrubbing or thelike. They can also be present e.g. as soil contaminants. The compoundsto be reduced can also be present in production lines, for producingdesired reduced compounds. The following reaction may apply:Ca₄+4A+2H₂O→4AH₂+CO₂wherein A is a hydrogen acceptor, and AH₂ may be replaced by equivalentsor subsequent conversion products. According to this embodiment,methane-oxidising bacteria to be used include those of the Thermotogalesorder as described above, as well as other methane-oxidisers, e.g. thoserelated to Desulfosarcina. The reduction step itself is in particular abiological reduction using suitable respiring organs. This process isschematically illustrated in FIG. 1.

In a particularly preferred embodiment, the anaerobic methane oxidationis used to reduce oxidised sulphur compounds, such as sulphate, sulphiteand elemental sulphur. In the following, reference is made to sulphate,but other sulphur-oxygen species, such as sulphite and hydrogenated(e.g. bisulphite) and neutral (e.g. sulphur trioxide) equivalents arealso comprised. The relevant total reaction can be simplified asfollows:CH₄+SO₄ ²⁻→HCO₃ ⁻+HS⁻+H₂OThis embodiment requires the presence of agents capable of transferringhydrogen equivalents to sulphate. Such agents are especiallysulphate-reducing micro-organisms, which are known in the art. Suitablesulphate-reducing micro-organisms include mesophilic and thermophilichydrogen-utilising stains from the bacterial sulphate-reducing genera,e.g. Desulforomonas, Desulfovibrio, Thermodesulfovibrio andDesulfotomaculum (e.g. the strain described in WO 98/02524) as well asthe archaeal sulphate-reducing genus, e.g. Archaeoglobus, such as A.profundus.

The conversion of sulphate by a coculture comprising the anaerobicmethane oxidisers as described above can be carried out in aconventional bioreactor having an inlet for sulphate-containing water,e.g. originating from a gas desulphurisation plant, a gas inlet formethane supply, a liquid outlet for sulphide-containing water, a gasoutlet for the resulting gas mixture containing e.g. residual methane,hydrogen, hydrogen sulphide, and optionally means for supporting thebiomass and for keeping it in effective contact with the liquid and(dissolved) gaseous materials, optional filters for separating gaseousproducts from the culture mixture and means for maintaining the desiredreactor temperature. Furthermore, a gas separation unit may be providedfor separating the resulting gas mixture and returning recovered methaneas well as a treatment unit for treating hydrogen sulphide, e.g. a unitfor biologically converting sulphide to elemental sulphur and forseparating off the sulphur. Since the bacteria do not use methane fortheir cell synthesis, further carbon sources, e.g. methanol, ethanol,organic acids, yeast extract or components thereof, or other organicmatter should be supplied to the bioreactor, in addition to methane.

Variations in the process of reducing sulphur compounds using acoculture of methane-oxidisers and sulphate-reducers are schematicallyillustrated in FIGS. 2-5. FIG. 2 is a flow diagram for reducing sulphateto sulphide, followed by biological oxidation of sulphide to elementalsulphur. FIG. 3 shows sulphate reduction in combination with metalprecipitation in the form of metal sulphides (MeS) by the hydrogensulphide produced and oxidation of the surplus hydrogen sulphide toelemental sulphur. FIG. 4 shows two variants of a process for producinghydrogen sulphide, either by separate stripping, or by hydrogen sulphideremoval using the methane stream. The hydrogen sulphide can beconcentrated and used for sulphuric acid production. FIG. 5 illustratessulphur dioxide removal from gases by scrubbing (first stage) followedby biological reduction as in FIG. 2.

In another embodiment of the process of the invention can be used forreducing noxious bromate or chlorate to less noxious bromide andchloride. These compounds can be present in process water from chemicalindustries. The reduction of bromate or chlorate requires the presenceof bromate- or chlorate reducing species, which can be sulphate-reducingbacteria and archaea. Species capable of reducing chlorate or bromatethose of the genera Dechlorosoma, Dechloromonas and Pseudomonas such asPseudomonas chloritidismutans, Dechloromonas agitata, Dechlorosomasuillum, strain GR-1. Similarly, nitrate reduction can be performedusing commonly known denitrifiers. Known denitrifying bacteria includePseudomonas stustzeri, Paracoccus denitrificans, Haloarcula marismortuiand Staphylococcus aureus.

According to a further embodiment, the process may be used for reducingmetal ions to their low-valence or zero-valence state. They can beprecipitated and separated in these lower valence states e.g. as oxides,hydroxides, carbonates, phosphates, sulphides or neutral metals. Thebiological reduction of metals is described for example in WO 00/39035.Examples of metals that can be reduced and converted to insoluble metalsor insoluble metal oxides, hydroxides or the like include selenium,tellurium, uranium, molybdenum, vanadium, chromium, and manganese.Bacteria capable of reducing these metals include the genera Geobacter,Pseudomonas, Shewanella, Desulfovibro, Desulfobacterium,Desulfomicrobium, Desulforomonas and Alteromonas. If desired, a movingsand filter can be used for separating the resulting metal precipitatesas described in WO 00/39035.

The use of the methane-oxidising bacteria in providing reducingequivalents in (biological) reduction processes is beneficial intechnical and economical terms. Current process using methane as theultimate reducing agent require the intermediary use hydrogen to beproduced from methane by chemical (catalytic) reforming. This impliesthe investment in and use of reformers or similar equipment and alsoconsumes about 50% of the methane by combustion needed to keep thecatalytic process at the necessary high temperature. These drawbacks areeliminated by the present biological process, thus resulting insubstantial cost savings both in equipment cost, and in operational cost(e.g. 50% lower methane consumption).

The process of the invention can be carried out in a conventionalbioreactor of the anaerobic type, having means for introducing a gasinto the reactor contents and means for carrying off gases from theheadspace of the reactor. The reactor can be of the stirred type, butpreferably the reactor is of a type having biofilms, present on carrierparticles such as sand, basalt, polymer particles etc., or in the formof granules, plates, membranes and the like, in order to optimisecontact between the substrate (methane) and the micro-organisms, and—incase of coculture—between the different microorganisms.

An example of a suitable reactor type for the biological conversion isthe so-called gaslift-loop reactor. This is a type of reactor which isespecially beneficial when a gaseous substrate has to be supplied forthe reaction. It is operated using a vertical circulation activated bythe gas (methane) introduced at the bottom of the reactor. An example ofsuch a reactor is the 500 m³ gaslift-loop reactor used at the zinc plantof Budel Zinc in the Netherlands. In this case 10 tons/day of sulphateis reduced biologically by addition of 12,000 nm³/day of hydrogen gas.Additionally, part of the bioreactor off-gas is recycled by compressorsto improve the efficiency of the hydrogen utilisation Here daily 20000nm³ of natural gas is converted in a steam reforming unit in order toprovide the required amount of hydrogen gas. When using anaerobicmethane oxidation as described above, the reformer a be by-passed andmethane can be introduced in the reactor directly.

Another suitable reactor type is a membrane bioreactor, wherein biomassretention is effected by passing the reactor effluent through a(membrane) filter. A membrane bioreactor can also be useful forseparating a gas product (such as hydrogen or hydrogen sulphide). Insuch as embodiment, a membrane which is permeable for the gas (e.g.hydrogen sulphide) separates reactor liquid from the gas space of thereactor, where an absorbing gas is passed for carrying away the gas.This allows low hydrogen sulphide levels to be maintained in the reactorwithout requiring a high water flow through the reactor.

The process of the invention can be carried out at atmospheric pressure,or—if desired—at elevated pressure, e.g. pressure in the of 10-100 bar,using appropriate pressure-resistant equipment. Elevated pressures mayhave the advantage of increasing the conversion rate of the biologicalprocesses using methane.

EXAMPLES

Strains. Thermotoga maritima (DSM 3109) and Archaeoglobus profundus (DSM5631) were purchased from the Deutsche Sammlung von Mikroorganismen undZellkulturen (Braunschweig, DE). T. lettingae (DSM 14385) andDesulfotomaculum sp. strain WW1 were isolated in our laboratory.

Cultivation techniques. Anaerobic culture techniques were usedthroughout this study. The cells were grown in an anaerobic mediumtypically supplemented with 0.15 g/l yeast extract. The medium contained(per liter demineralised water) 0.335 g of KCl, 4.0 g of MgCl₂. 6H₂O,3.45 g of MgSO₄. 7H₂O, 0.25 g of NH₄Cl, 10 g of NaCl, 0.10 g of K₂PO₄,4.0 g of NaRCO₃, 0.5 g of Na₂S. 9H₂O, 1.0 g of Na₂SO₄, 0.73 g ofCaCl₂.2H₂O, 10 ml of trace element and 5 ml of two-times concentratedvitamin solution which were based on medium 141 of DSM(http://www.dzmz.de). The medium was boiled and cooled to roomtemperature under a stream of O₂-free N₂ gas. The medium wasanaerobically dispensed into serum bottles and a gas phase of 180 kPaN₂/CO₂ (80/20, v/v) was applied. The bottles were closed with butylrubber stoppers and sealed with crimp seals. The medium was autoclavedfor 20 min at 121° C. Stock solutions of NaHCO₃, Na₂S, CaCl₂ and vitaminsolutions wee prepared under a nitrogen gas atmosphere and added aftersterilisation. The vitamin solution contained higher amounts ofvitamin-B₁₂ and vitamin-B₁ resulting in concentrations in the medium of0.5 and 0.1 mg. 1⁻¹, respectively. Thiosulfate was added from 1 Mfilter-sterilized stock solution. Calculated amounts of ¹³C-methane wereinjected into bottles prior to autoclaving. For coculture experiments,A. profundus and Desulfotomaculum sp. strain WW1 were grown on H₂ andCO₂ in 248 ml serum vials with 50 ml medium at 80° C. and 65° C.,respectively. As additional carbon source, 1 mM of sodium acetate wasadded. The sulphate reducers were grown for 1 day and then the gas phasewas changed to 180 kPa N₂/CO₂ (80/20, v/v) and CH₄ gas was added to thefinal concentration of 1.75 mmol per vial. The A. profundus culture wasinoculated with T. maritima and the Desulfotomaculum sp. with T.lettingae. For inoculation, methane adapted cultures of T. maritima andT. lettingae were used. Since the medium already contained nearly 1 mmolof sulphate per vial, no sulphate was added.

Analytical techniques. Highly pure (min 99% ¹³C) methane gas wasobtained from Campro Scientific B.V. (Veenendaal, NL). Serum vials wereprepared with approximately 1.75 mmol of ¹³C-methane as substrate in 50ml medium. Control bottles were prepared with or without ¹³C-methane,thiosulphate and organisms. NMR-tubes contained the sample, 10% (v/v)D₂O and 100 mM dioxane to give a final volume of 15 ml. Theproton-decoupled ¹³C-NMR-spectra of the samples were recorded at 75.47MHz on a Bruker AMX-300 NMR spectrometer. For each spectrum 7200transients (5 h) were accumulated and stored on disc using 32 k datapoints, a 45° pulse angle and a delay time of 1 s between pulses. Themeasuring temperature was maintained at 10° C. and the chemical shiftbelonging to the dioxane carbon nuclei (67.4 ppm) was used as aninternal standard. The deuterium in the samples (10%, v/v) was used forfield lock and dioxane as an internal standard.

Hydrogen and methane were determined at room temperature by either gaschromatography (GC) (see: Stams et al. Appl. Environ. Microbiol. (1993)59 114-1119) or GC (Hewlett Packard model 5890) equipped with a massselective detector (MS). Methane, carbon dioxide and their stableisotopes were separated on a capillary column (innowax, 30 m×0.25 mm(df=0.5 μm), Packard, NL) with helium as the carrier gas. Gas samples(200 μl) were injected in a split injector (inlet pressure 1 kPa; splitratio 25:1) at a column temperate of 35° C. Methane and carbon dioxideand their stable isotopes were monitored at m/z 16 and 17, and 44 and45, respectively. Total methane and CO₂ concentrations were determinedquantitatively by gas chromatography (see Stams et al. (1993 above). TheH¹³CO₃ ⁻ concentration in the liquid phase was calculated from theamount of CO₂ which accumulated in the gas phase after acidification.Thiosulphate and sulphate were analysed by HPLC (see: Scholten andStams, Antonie van Leeuwenhoek (1995) 68, 309-315). Sulphide wasdetermined as described by Trüper and Schlegel (see: Antonie vanLeeuwenhoek (1964) 30, 225-238).

Example 1 Anaerobic Methane Oxidation by Thermotoga Species

Thermotoga maritima and T. lettingae were incubated with highly pure¹³C-labelled methane under strictly anaerobic conditions in the presenceof thiosulphate as the electron acceptor. The experiments were carriedout was studied in duplicate cultures at incubation temperatures of 80°C. and 65° C., respectively. Methane oxidation and product formationwere determined by analysing the gas phase by gas chromatography (GC)and GC-mass spectroscopy (MS) and by using Nuclear Magnetic Resonancespectroscopy (NMR) for liquid samples.

In acidified samples, the total CO₂ which accumulated in the gas phaseafter 40 days of incubation was measured by using GC (Table 1). In thecontrol incubation without methane which contained totally 4.87 mmol ofCO₂ per vial, slight growth was observed due to the presence of yeastextract in the medium and consequently, unlabelled CO₂ was found. Growthin the presence of ¹³C-methane and thiosulphate by T. maritima and T.lettingae resulted in significantly increased cell numbers (Table 1).The stoichiometry of methane conversion by the two cultures yieldednearly equal amounts of products after 40 days of incubation. Thepercentage of ¹³C in CO₂ was 5.9% in liquid phase and 2.1% in gas phasefor T. lettingae. These values for T. maritima were 6.2% and 2.4%,respectively. The ratio of methane oxidation to carbon dioxide andthiosulphate reduction to sulphide in both samples were approximately1:1 and 1:2, respectively. Nearly 1 mmol of ¹³C-metane per vial wasutilised by both bacteria. The rest of the methane in the vials was notutilised even after 40 days of prolonged incubations. However, when lessmethane was added (up to 0.5 mmol per vial), all ¹³C-methane wascompletely utilised by the two bacteria. In both cases, measurablemethane conversion started after around 10 days. The rates of anaerobicmethane transformation by T. maritima and T. lettingae were 32 and 30μmol per vial per day, respectively. The ¹³C-carbon recoveries for T.maritima and T. lettingae were calculated to be 82% and 79%,respectively. TABLE 1 a ¹³C-methane oxidation in the presence ofthiosulphate by T. maritima‡. Total Number of Thio- Total SulphideHydro- cells ¹³CH₄ sulphate CO₂* # gen (ml)† Day 0 1.75 0.99 4.87 0.050.00 4 × 10⁵ Day 40 0.68 0.00 5.75 1.96 0.05 8 × 10⁷ Utilised/ 1.07 0.990.88 1.91 0.05 produced TABLE 1 b ¹³C-methane oxidation in the presenceof thiosulphate by T. lettingae‡. Total Number of Thio- Total SulphideHydro- cells ¹³CH₄ sulphate CO₂* # gen (ml)† Day 0 1.75 1.01 4.87 0.050.00 5 × 10⁵ Day 40 0.71 0.00 5.68 1.39 0.04 1 × 10⁷ Utilized/ 1.05 1.010.81 1.84 0.04 produced‡The values were calculated in mmol per vial and corrected with thecontrol samples. All measurements were done in duplicates and thehighest values of each sample were calculated*Total CO₂ includes CO₂ from the medium composition in liquid and gasphases and ¹³C-carbon dioxide formed#Total sulphide includes sulphide from the medium composition in liquidand gas phases and sulphide formed during methane oxidation†Ballooning cells were not taken into account during counting of thecells

Example 2 Biomass Analysis

Thermotoga lettingae and Thermotoga martima were grown with labelledmethane and thiosulfate at 65 and 80° C., respectively. After growth,cells were centrifuged. The percentage ¹³C in the supernatant and thecell pellet were analysed. As shown before, ¹³C labelled bicarbonate wasformed, but we could not detect any label incorporation in biomass. Thisindicates that no cell biomass is formed from methane or its degradationproducts, but from yeast extract supplied to the medium. Thus, it seemsthat the bacteria have a split metabolism.

Enzyme activity: Methane-oxidizing activity was determined in cell freeextracts prepared from methane grown cells. We could measure highactivities of an NAD-dependent methane dehydrogenase (>1□mol.min⁻¹.mg⁻¹protein) at pH 9 and 65° C. At 80° C. no activity couldbe measured. The apparent reaction is:Methane+NAD⁺+H2O→methanol+NADH+H⁺

The Gibbs free energy change of this reaction is about +90 kJ per mol.Therefore, this reaction can only occur when the concentration of thesubstrates are high and the concentration of the products low. We couldshow that the reaction is inhibited by addition of methanol to the assaymixture and that a higher activity was measured when the NAD⁺concentration was increased.

Example 3 Sulphate Reduction by Coculture with Thermotoga Species

Coculture experiments were performed by growing Archaeoglobus profunduswith T. maritima at 80° C. and Desulfotomaculum sp. strain WW1 with T.lettingae at 65° C. (Table 2). In both cases, sulphate was utlised asthe electron acceptor. The ratio of CO₂ formation from methane andsulphate conversion to sulphide were nearly 1:1 for both cocultures. Thecalculated rates of methane conversion were slower than in the case withthiosulphate, which was about 20 μmol per vial per day. Conversion ofmethane coupled to sulphate reduction led to more than ten-foldincreased cell numbers of both the Thermotoga species and thesulphate-reducing microorganisms. The ¹³ C-carbon recoveries for thecocultures of T. maritima and T. lettingae were calculated to be 85% and78%, respectively, TABLE 2a ¹³C-methane oxidation by T. maritima (T.m.)in coculture with Archaeoglobus profundus (A.p.) in the presence ofsulphate‡. Total Number of Total Total Sulphide cells ¹³CH₄ SulphateCO₂* # (ml)† Day 0 1.75 1.10 4.87 0.11 3 × 10⁵ (T.m.) 2 × 10⁶ (A.p.) Day40 1.07 0.31 5.53 0.85 5 × 10⁷ (T.m.) 7 × 10⁸ (A.p.) Utilised/ 0.68 0.790.57 0.74 produced TABLE 2b ¹³C-methane oxidation by T. lettingae (T.l.)in coculture with Desulfotomaculum sp. strain WW1 (WW1) in the presenceof sulphate‡. Total Number of Total Total Sulphide cells ¹³CH₄ SulphateCO₂* # (ml)† Day 0 1.75 1.10 4.87 0.07 3 × 10⁶ (T.l.) 3 × 10⁶ (WW1) Day40 1.21 0.50 5.28 0.53 2 × 10⁷ (T.l.) 8 × 10⁸ (WW1) Utilised/ 0.54 0.600.41 0.51 producedFor symbols see TABLE 1.

Previous results showed that T. maritima and T. lettingae are able togrow on other C₁-compounds like H₂—CO₂, formate, methanol, andmethylamine in the presence thiosulphate and yeast extract¹⁸. We alsoobserved that addition of 0.5 to 5.0 g/l of yeast extract resulted inbetter growth and consequently slightly faster methane oxidation than inthe original medium. In the absence of yeast extract, the pure culturescould not grow, even not on glucose. The rate of methane utilisationcould be increased from 32 to 33 μmol per vial per day for T. maritimaand from 30 to 31 μmol per vial for T. lettingae when 2.5 g/l yeastextract was added in the medium. However, similarly to the findingswithin original medium which contained 0.15 g/l of yeast extract,methane oxidation started only after around 10 days of incubation. Whenmore than 5.0 g/l of yeast extract was added, growth for both organismswas better but methane oxidation rates were not higher than the obtainedvalues.

1. A process for converting methane to produce hydrogen or hydrogenequivalents, characterised in that methane is subjected anaerobically tothe activity of methane-oxidising bacteria of the order of theThermotogales.
 2. A process according to claim 1, wherein themethane-oxidising bacteria comprise a Thermotoga species.
 3. A processaccording to claim 2, wherein the Thermotoga species comprises T.maritima or T. lettingae.
 4. A process according to any one of claims1-3, which is carried out at a temperature between 25 and 90° C.
 5. Aprocess according to any one of claims 1-4, which is carried out in thepresence of thiosulphate.
 6. A process for reducing chemical compoundsby biological reduction using hydrogen equivalents, characterised inthat the hydrogen equivalents are produced by subjecting methane toanaerobic methane-oxidising bacteria of the order of the Thermotogales.7. A process according to claim 6, wherein sulphur compounds are reducedto sulphide using a sulphate-reducing species.
 8. A process according toclaim 7, wherein the sulphur compounds comprise sulphate and/orsulphite.
 9. A process according to claim 7 or 8, wherein the anaerobicmethane-oxidising species comprises a Thermotoga, Thermosipho orFervidobacterium species.
 10. A process according to claim 7 or 8,wherein the sulphate-reducing species comprises an Archaeglobus,Desulfotomaculum, Desulforomonas, Desulfovibrio or Thermodesulfovibriospecies.
 11. A process according to claim 6, wherein metals are reducedfrom a high valence state to a low-valence or zero-valence state.
 12. Aprocess according to any one of claims 6-11, wherein a temperature ofbetween 25 and 90° C. is used.
 13. A mixed culture, containing one oremore anaerobic methane-oxidising Thermotogales species, and one or moresulphate-reducing or metal-reducing Archaeglobus, Desulfotomaculum,Desulforomonas or Desulfovibrio species.